The enzyme-linked immunospot (ELISPOT) assay has advanced into a useful and widely applicable tool for the evaluation of T-cell responses in both humans and animal models of diseases and/or vaccine candidates. Using synthetic peptides (either individually or as overlapping peptide mixtures) or whole antigens, total lymphocyte or isolated T-cell subset responses can be assessed either after short-term stimulation (standard ELISPOT) or after their expansion during a 10-day culture (cultured ELISPOT). Both assays detect different antigen-specific immune responses allowing the analysis of effector memory T cells and central memory T cells. This paper describes the principle of ELISPOT assays and discusses their application in the evaluation of immune correlates of clinical interest with a focus on the vaccine field. 1. Introduction The ELISPOT assay was originally developed to detect antigen-specific antibody-secreting B cells [1] and then evolved for the enumeration of antigen-specific cytokine-secreting T cells [2]. Since then the ELISPOT assay has been used to measure antigen-specific T cells by detecting cytokines secreted by T cells after stimulation with synthetic peptides (used as a pool or as single peptides) or whole antigens (such as proteins or lysates). The ELISPOT assay not only permits the quantification of T cells that respond to a specific antigen but it also allows the detection of functionally relevant molecules upon specific stimulation of effector T cells. Although the tetramer staining method provides valuable information regarding the frequency of T cells (particularly CD8+ T cells), it needs to be combined with intracellular cytokine staining for the functional evaluation of T cells. However, the tetramer staining approach requires knowledge of the relevant epitope(s) and its restricting MHC molecule, limiting the use of this approach to the clinical setting. Thus, the ELISPOT is an attractive alternative because it is not limited by HLA restriction. Additionally, the low number of cells required to accurately assess T-cell activity (roughly tenfold less cell material than flow cytometry-based assays) as well as the high sensitivity and specificity of the assay makes the ELISPOT an optimal method for clinical monitoring [3], especially in clinical settings when the number of patient cells is limited (e.g., immunosuppressed subjects or pediatric patients). However, ELISPOT does not allow phenotypic characterization of antigen-stimulated T cells. Thus, magnetic isolation or depletion of T-cell subsets from peripheral blood mononuclear
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